The present invention relates generally to methods for the synthetic preparation of peptides, and more particularly to the preparation of peptides of improved activity and complex structure, and to the products, including bioactive agents, pharmaceutical compositions and vaccines, that may embody such constructs.
The synthesis of peptides or proteins has become highly efficient with the advances of the solid-phase peptide synthesis and recombinant DNA technology. Solid-phase peptide synthesis with the aid of automation and other mechanical devices can quickly produce a peptide of greater than 100 amino acids or a library of hundreds of short peptides. Likewise, recombinant DNA technology with an optimal expression system can produce proteins accurately and in large quantity. The development of a method of chemical ligation of peptide segments would be desirable, as it would incorporate both the efficiency of the solid-phase method to generate specific segments, and the availability of proteins generated by the recombinant method. The combination of the two types of production of peptide segments would enable engineered proteins to contain unusual structures or nongenetic encoded amino acids by a specific ligation method.
A strong impediment to the development of such an approach has been a lack of an efficient method for their synthesis. In particular, there is no effective chemical method to selectively couple two unprotected peptide segments to form an amide bond. In general, protecting groups are necessarily attached to nontarget functional groups on the first peptide segment prior to activation of the C-.alpha. of the carboxylic group by a coupling reagent and the consequent peptide bond formation with the N-.alpha. of the amino group of the second protected peptide segment. As a result, the development of various protecting group schemes has been the key for the conventional approach of ligating peptide segments.
However, the use of protected peptide segments is incompatible with the overall scheme of engineering proteins using proteins produced by means of recombinant DNA technology as a source. Such technique is also limited as it is labor-intensive and unpredictable, partly due to the solubility and coupling difficulties of protected peptide segments. Often, large protected peptide segments are minimally soluble in even the most powerful polar aprotic solvents such as dimethylsulfoxide (DMSO) and dimethylformamide (DMF).
The problem of the insolubility of protected peptide segments has been addressed with limited success in several ways, including the use of (1) a partial protecting group strategy which masks all side chains except those of Ser, Thr, and Tyr, and (2) a minimal protecting group strategy which masks only thiol and amino side chains. Protecting groups used in all these approaches alter peptide conformations. This creates a difficult problem in the synthesis of large peptides, since folding and renaturation are required after the completion of the synthesis and removal of the protecting groups. These limitations, coupled with the ease of obtaining proteins and protein domains through recombinant DNA technologies, have suggested the need to develop a new strategy for ligating unprotected peptides and proteins in order to engineer new proteins with unusual structures, architectures and functions.
Since protecting groups are the root of the problem, scientists have developed two ligation strategies in the past ten years which use unprotected segments. One of the methods requires the use of enzymes in the reverse proteolysis process in conjunction with a high content of water-miscible solvents. Although enzymatic synthesis has been successful with small peptides, enzymatic synthesis of large peptides has presented difficulties. The stringent criteria demanded by using high molar concentrations of peptide segments accompanied by rapid completion of the reverse proteolytic process without the attendant hydrolysis or transpeptidation have been prohibitive obstacles in the enzymatic synthesis of large peptides. Nevertheless, the use of enzymes in coupling unprotected peptide segments eliminates the necessity of activating the carboxylic group involved in the coupling reaction of the peptide segments. Furthermore, it also provides the ability to perform the reaction in an aqueous environment.
Another strategy uses a tricyclic aromatic template containing an aryl alcohol and a thiol to form an active ester with the carboxyl segment and a disulfide with the amino segment, respectively, in order to bring two unprotected peptide segments in close proximity with each other. Such positioning of the peptide segments enables them to undergo an O to N-acyl transfer reaction (Fotouhi, N. et al., 1989; Kemp, D. S. et al., 1991).
A problem with the currently accepted methods of protein synthesis which include both conventional liquid state and solid state peptide syntheses is that their application is limited to small straight chain peptide segments, whereas the need exists for such a method of synthesis to be available for long straight chain peptides, branched straight chain pep tides and circular peptides.
From the foregoing, it is apparent that a need exists for the development of an efficient and reliable strategy for peptide synthesis that facilitates the preparation of peptides and proteins without limitation as to their size and structural complexity, that results in the formation of stable and desirably active molecules. It is toward the fulfillment of this need that the present invention is directed.